1. Field of the Invention
The present invention relates generally to assays and more specifically to light emission- or absorbance-based binding assays.
2. Background Art
Binding assays, for example, immunoassays and receptor based assays, are widely used in the medical community as diagnostic tests. There are several binding assays that have been produced and are currently on the market since the principle was developed by R. S. Yalow and S. A. Berson, J. Clinical Investigations, 39 1157(1960). An example of a binding assay is Radioimmunoassay (RIA) (D. Monroe, Anal. Chem., 56 920A(1984)). All immunoassays exploit the binding capabilities of antibodies. However other molecules that are capable of recognizing and specifically binding other molecules may be employed. Antibodies are protein molecules which are frequently considered fighters of infections. They fight infections by binding to the infectious material in a specific manner, forming a complex. This is then a signal to the organism to reject that complex. However, antibodies may also be produced to bind to an individual compound, as a key fits a lock. To be useful in an assay, this recognition event must generate a signal that is macroscopically observable. The method employed to generate such a signal is what distinguishes the various types of immunoassays. In the above example, radioactivity is employed. RIA is quite sensitive and widely used, but the expense and restrictions for handling radioactive material makes alternative immunoassays desirable.
Fluorescence and chemiluminescence have been used in various types of assays, such as enzyme assays and immunoassays. In each of these systems, energy-coupling reactions have been exploited.
Carmel et al., FEBS Letters, Vol. 30, No. 1, February 1973, pages 11 through 14, describe the use of fluorescent donors and acceptors which are in close proximity to each other to measure the rate of enzymatic cleavage of a suitable labelled peptide. In their system, a peptide is labelled with two fluorophores. One fluorophore (the donor) accepts excitation light and fluoresces. If the other fluorophore (the acceptor) is in close proximity to the donor, it can accept the emitted light of the donor as excitation light or energy and then emit its own fluorescence. Since Forster, Ann. Physik., 2 (1948) 55, has shown that the probability of the donor exciting the acceptor decreases with the sixth power of the distance between them, if they are separated by enzymatic cleavage of the peptide linker, the fluorescence of the acceptor will decrease substantially. Thus, a measure of the fluorescent intensity of the acceptor is inversely proportional to the rate of enzymatic activity. Although such a system is quite sensitive, it is difficult to find appropriate donors and acceptors such that the donor may be exclusively excited by the incident radiation without exciting the acceptor.
Binding assays have been produced by using the donor/acceptor scheme described above. In this case, the donor is a fluorescently labelled hapten and the acceptor is the antibody with many fluorescent acceptors attached. This large concentration of fluorescent acceptors is needed because the distances are greater than in simple peptide enzymatic substrates. However, the same problems occur with finding appropriate donors and acceptors that occur with enzymatic substrates. Patel et al., Clin. Chem., Vol. 29, No. 9, 1983, 1604-1608, have overcome some of these difficulties by using chemiluminescence to excite the acceptor. To achieve the reported high sensitivities, a very sensitive instrument must be employed.
Similar systems (Pohl et al., Analytical Biochemistry 165, 96-101 (1987)) have used fluorescent quenching to measure the distance between a quencher and a fluorophore, both attached to the same peptide linker. The increase in fluorescence when the peptide linker is cleaved is a measure of the enzymatic activity. However, the quencher is not very efficient in reducing fluorescent such that only a five- to eight-fold increase in fluorescence is observed when the peptide linker is cleaved.
Among the numerous binding assays that are possible, the one of most interest is that for detection of small molecular weight species. For immunoassays, i.e., use of an antibody as the binding molecule, this type of binding assay is termed a competition immunoassay.